Processing microtitre plates for covalent immobilization chemistries

ABSTRACT

Disclosed herein is a method of: treating an organic polymer with an electron beam-generated plasma; exposing the treated polymer to air or an oxygen- and hydrogen-containing gas, generating hydroxyl groups on the surface of the polymer; reacting the surface with an organosilane compound having a chloro, fluoro, or alkoxy group and a functional or reactive group that is less reactive with the surface than the chloro, fluoro, or alkoxy group; and covalently immobilizing a biomolecule to the functional or reactive group or a reaction product thereof.

This application is a divisional application of pending U.S. Pat. No.9,962,676 issued on May 8, 2018, which is a divisional application ofpending U.S. Pat. No. 9,182,392 issued on Nov. 10, 2015, which is adivisional application of U.S. Pat. No. 8,651,158 issued on Feb. 18,2014, which claims the benefit of U.S. Provisional Application No.61/261,843, filed on Nov. 17, 2009. The provisional application and allother publications and patent documents referred to throughout thisnonprovisional application are incorporated herein by reference.

TECHNICAL FIELD

The present disclosure is generally related to microtitre plates forassays.

DESCRIPTION OF RELATED ART

Microtitre plates are effective solid-phase platforms for multiplexed,high-throughput screening and analysis of biomolecule interactions. Thismulti-well format, available in 6, 24, 96, 384, 1536, and even 3456 or9600-well plates, is widely used in both industry and medical fields dueto its ease of automation, high capacity for paralleled data collection,and versatile application of technologies (e.g., proteomics, functionalgenomics, biomolecule separation and purification). Microtitre platesare manufactured using a variety of polymeric materials (e.g.,polypropylene, polycarbonate), but polystyrene is most commonly employedbecause it readily adsorbs (via non-covalent interaction) proteins, hasexcellent optical as well as mechanical properties, and iscost-effective. However, several of these intrinsic properties ofpolystyrene also present disadvantages: poor chemical resistance,difficulty controlling surface chemistry, protein denaturation,desorption and/or loss of biomolecule activity (Rebeski et al.,“Identification of unacceptable background caused by non-specificprotein adsorption to the plastic surface of 96-well immunoassay platesusing a standardized enzyme-linked immunosorbent assay procedure” J.Immunological Methods, 226, 85-92 (1999). Indeed, it has been estimatedthat less than 10% of adsorbed protein molecules retain their activity(Butler, “Solid Supports in Enzyme-Linked Immunosorbent Assay and OtherSolid-Phase Immunoassays” Methods, 22(1), 4-23 (2000)).

As a consequence of the structural complexity and diversity ofbiological molecules and the highly specific recognition mechanism forbiomolecule capture, surface attachment design must take intoconsideration native conformation, molecular orientation, andcross-reactivity (Kusnezow et al., “Solid supports for microarrayimmunoassays” Journal of Molecular Recognition, 16(4), 165-176 (2003)).Although the simplest means of biomolecule immobilization is throughsurface adsorption, a non-covalent binding event that takes place onmost microtitre plates, covalent attachment schemes represent a morerobust approach that can ensure functional display of the biomolecule ofinterest (Goddard et al., “Polymer surface modification for theattachment of bioactive compounds” Progress in Polymer Science, 32(7),698-725 (2007)). Different surface modification strategies havepreviously been reported for the covalent attachment of biomolecules topolymeric substrates (e.g., poly(methyl methacrylate) (PMMA),polydimethysiloxane (PDMS), polystyrene (PS), polyvinylidenedifluoride(PVDF), etc.). But generally, these studies relied on variable thicknesscoatings of amine-bearing copolymers and biopolymers (Bai et al.,“Surface Modification for Enhancing Antibody Binding on Polymer-BasedMicrofluidic Device for Enzyme-Linked Immunosorbent Assay” Langmuir,22(22), 9458-9467 (2006); Boulares-Pender et al.,“Surface-functionalization of plasma-treated polystyrene byhyperbranched polymers and use in biological applications” Journal ofApplied Polymer Science, 112(5), 2701-2709 (2009)), inert proteins(Eteshola et al., “Development and characterization of an ELISA assay inPDMS microfluidic channels” Sensors and Actuators B: Chemical, 72(2),129-133 (2001)), metals (Darain et al., “On-chip detection of myoglobinbased on fluorescence” Biosensors and Bioelectronics, 24(6), 1744-1750(2009)) or sol-gel matrices (Wang et al., “Microfluidic immunosensorbased on stable antibody-patterned surface in PMMA microchip”Electrochemistry Communications, 10(3), 447-450 (2008)) applied throughphysical or chemical vapor deposition for covalent bioimmobilization.Ideally, surface modification approaches should aim to introduce no morethan a single monolayer of a desired functional group to a substratesurface, yet in these cases, bioimmobilization has little to do with thecore substrate but rather the deposited film or coating. The challengeof polymer surface engineering is underscored by the limited number ofcommercially available polymeric substrates (e.g., microtitre plates)that possess the correct surface characteristics to facilitate a direct,covalent biomolecule attachment.

In recent years, there has been an emerging interest in the applicationof self-assembled monolayers for bioimmobilization on solid support.Silane coupling chemistry is highly selective and allows thefunctionalization of a substrate surface with different functionalgroups (e.g., epoxy, amine, thiol, aldehyde, etc.). Organosilanes havebeen used to covalently attach biological molecules onto inorganicsubstrates such as silica (e.g., glass, silicon via siloxy linkage) andmetal (e.g., gold, copper, silver via thiol linkage) (Plueddemann, Ed.,Silane coupling agents. New York, Plenum Press (1991); Mittal, Ed.,Silanes and other coupling agents. The Netherlands, VSP BV (2000)).There has been significantly less research on surface modification usingsilane monolayers on polymers. Polymer surface modification techniques(e.g., ion beam treatment, plasma treatment, UV/ozone treatment) havebeen reported to create pendant surface hydroxyl groups on polymericsurfaces, which allows the application of the same silanizationchemistry used for inorganic substrates (Inagaki et al., “SurfaceModification of PET Films by Pulsed Argon Plasma” J. Appl. Polym. Sci.,85, 2845-2852 (2002); Kawase et al. “End-capped fluoroalkyl-functionalsilanes. Part II: Modification of polymers and possibility ofmultifunctional silanes” Journal of Adhesion Science and Technology, 16,1121-1140 (2002); Cheng et al., “Direct-write laser micromachining anduniversal surface modification of PMMA for device development” Sensorsand Actuators B: Chemical, 99(1), 186-196 (2004); Prissanaroon et al.,“Fabrication of patterned polypyrrole on fluoropolymers for pH sensingapplications” Synthetic Metals, 54, 105-108 (2005); Long et al.,“Water-Vapor Plasma-Based Surface Activation for TrichlorosilaneModification of PMMA” Langmuir, 22, 4104-4109 (2006)). However, in theseprevious studies, the organosilanes lacked reactive end-functionalitiesand were deposited on the polymeric surface primarily as a means ofmodulating surface properties (e.g., adhesion, wettability). Polystyrenecan be chemically treated to support silane-dependant attachment ofbiomolecules in enzyme-linked immunosorbent assay (Kaur et al.,“Strategies for direct attachment of hapten to a polystyrene support forapplications in enzyme-linked immunosorbent assay (ELISA)”, AnalyticaChimica Acta, 506(2), 133-135 (2004)), but as with most wet methods forsurface modification, this approach requires extensive treatment incorrosive solutions that result in the non-specific biomoleculeattachment, irregular surface etching and the production of a wide rangeof oxygen containing functional groups (e.g., C═O, O—C═O, CO₃) andtherefore less useful for applications that require more specificfunctionalities.

In addition to the surface attachment mechanism, three-dimensionalsurface structures, as those created by chemical and physical etchingtechniques or thin film deposition, also play a significant role inmodulating efficacy of bioimmobilization and functionality of thebiointerface. Surface morphology has been shown to affect the activityof the immobilized biomolecule, specific and non-specific binding ofmacromolecules and cells, as well as surface energy of the coresubstrate (Cretich et al., “A new polymeric coating for proteinmicroarrays” Analytical Biochemistry, 332(1), 67-74 (2004); Chiari etal., “Peptide microarrays for the characterization of antigenic regionsof human chromogranin A” Proteomics, 5(14), 3600-3603 (2005); Tuttle etal., “Influence of biologically inspired nanometer surface roughness onantigen—antibody interactions for immunoassay—biosensor applications”Int. J. Nanomedicine, 1(4), 497-505 (2006)). Surface roughness cansignificantly alter the biophysical and biochemical behavior of thebiointerface (Lamb et al., “Enzyme immobilisation on colloidal liquidaphrons (CLAs): the influence of protein properties” Enzyme andMicrobial Technology, 24(8-9), 541-548 (1999); Wentworth et al.,“Application of chitosan-entrapped beta-galactosidase in a packed-bedreactor system” Journal of Applied Polymer Science, 91(2), 1294-1299(2004)). When surface roughness and surface chemistry are both altered,as it is typically the case for most surface modification methods, it isdifficult to identify the contribution of each variable to specific andnon-specific biomolecule attachment and activity. Therefore, advancedsurface engineering is necessary to provide precise, independent controlof chemical functionality (i.e., density and type of reactive moieties)and surface topology (i.e., surface roughness) down to the nanometerscale.

BRIEF SUMMARY

Disclosed herein is a method comprising: treating an organic polymerwith an electron beam-generated plasma; exposing the treated polymer toair or an oxygen- and hydrogen-containing gas; reacting the surface withan organosilane compound having a chloro, fluoro, or alkoxy group and afunctional or reactive group that is less reactive with the surface thanthe chloro, fluoro, or alkoxy group; and covalently immobilizing abiomolecule to the functional or reactive group or a reaction productthereof. The treating and exposing generates hydroxyl groups on thesurface of the polymer.

Also disclosed herein is an article comprising: a polymeric microtitreplate comprising a plurality of wells and two or more differentorganosilane compounds having a functional or reactive group and boundto the surfaces of the wells. At least two of the wells have differentorganosilane compounds bound therein

Also disclosed herein is an article comprising: a polymeric microtitreplate comprising a plurality of wells and one or more organosilanecompounds bound to the surfaces of the wells. The organosilane compoundsin at least two different wells are each reacted with a differentcrosslinker.

Also disclosed herein is a method comprising: treating an organicpolymer microtitre plate with an electron beam-generated plasma; andexposing the treated polymer to air or an oxygen- andhydrogen-containing gas. The treating and exposing generates hydroxylgroups on the surface

BRIEF DESCRIPTION OF THE DRAWINGS

A more complete appreciation of the invention will be readily obtainedby reference to the following Description of the Example Embodiments andthe accompanying drawings.

FIG. 1 schematically illustrates an example plasma-generating apparatus.

FIGS. 2A-D show bioimmobilization assays. FIG. 2A shows a plasma-treatedplate incubated with MPTES. FIG. 2B shows a non-plasma-treated plateincubated with MPTES. FIG. 2C shows a plasma-treated plate incubatedwith APTES. FIG. 2D shows a non-plasma-treated plate incubated withAPTES. Microfluor I (NUNC) polystyrene microtitre plates were treatedusing sample C7 conditions (Table 3). Plasma-treated plates (A and C)and non-plasma-treated plates (B and D) were then incubated with thiol-or amine-terminated silanes (specifically, 3-mercaptopropyl (MPTES) (Aand B) and 3-aminopropyl tri(m)ethoxy silanes (APTES) (C and D))followed by incubation with a thiol specific GMBS or amine specific BS3crosslinker. The plates were rinsed with water and solutions containingfluorescently-labeled antibodies were added to wells. Plates werefurther incubated in PBS containing 0.05% Tween-20 to remove adsorbedproteins and measured for bound fluorescence, indicating the covalentattachment of fluorescent protein. Multiple controls were included:±plasma treatment, ±APTES and MPTES, ±GMBS and BS3, and differentconcentrations of fluorescent protein.

FIG. 3A shows an XPS survey spectrum of a plasma-treated microtitreplate using the optimum conditions discussed herein and FIG. 3B shows anXPS survey spectrum of an untreated microtitre plate.

FIG. 4A shows an XPS high resolution spectrum of the C1s peak for aplasma treated microtitre plate and FIG. 4B shows an XPS high resolutionspectrum of an untreated microtitre plate.

FIG. 5A shows an AFM image of an untreated microtitre plate, FIG. 5Bshows an AFM image of an untreated silanized microtitre plate, FIG. 5Cshows an AFM image of a plasma-treated microtitre plate, and FIG. 5Dshows an AFM image of a plasma-treated silanized microtiter plate.

FIG. 6 shows immobilization schemes using aminosilane (left) ormercaptosilane (right).

FIG. 7 shows binding of Cy3-LPS by antimicrobial peptides (cecropin A,melittin, cecropin A-melittin hybrid, and cecropin P1) immobilized ontosilanized glass slides (white bars), plasma-modified microtiter plates(via MPTES and GMBS, gray bars), and commercial amine-directed,preactivated plates (striped bars). As two instruments were used toobtain and extract fluorescence data (microtiter plate reader, confocalmicroarray scanner), all values were normalized to those obtained withmelittin.

FIG. 8 shows fluorescent intensities of Cy3-IgG (10 μg/mL)immobilization to plasma-treated Microfluor I (covalent immobilizationfollowing Scheme Two), Microfluor I (physisorption), Pierce ReactibindMaleic Anhydride, and NUNC Immobilizer Amino (covalent immobilizationaccording to manufacturers' instructions); gray bars=no detergent wash,white bars=detergent wash.

FIG. 9 shows biomolecule immobilization efficacy as a function of plasmatreatment time.

DETAILED DESCRIPTION OF EXAMPLE EMBODIMENTS

In the following description, for purposes of explanation and notlimitation, specific details are set forth in order to provide athorough understanding of the present disclosure. However, it will beapparent to one skilled in the art that the present subject matter maybe practiced in other embodiments that depart from these specificdetails. In other instances, detailed descriptions of well-known methodsand devices are omitted so as to not obscure the present disclosure withunnecessary detail.

Microtitre plates are now a standard tool in analytical research andclinical diagnostic testing laboratories, most specifically forenzyme-linked immunosorbent assays (ELISAs). The most commonly usedmaterial for the production of microtitre plates is polystyrene, due toits clarity when used unadulterated and its propensity to adsorbbiomolecules of interest, such as antibodies and other proteins.Although attachment through adsorption is simple and easy, biomoleculesimmobilized in this way may desorb under a variety of conditions, andtheir activity and/or presentation may be compromised by the randomorientation by which they are adsorbed. Additionally, many biomoleculescannot be reproducibly immobilized in functional form on standardpolystyrene microtitre plates. For this reason, microplates capable ofcovalent attachment of biomolecules have been developed and are nowcommercially available. These include maleic anhydride- andmaleimide-activated microplates (Thermo Scientific) and ImmobilizerAmino plates (Nunc). Although useful in many settings, these plates aresignificantly more expensive than standard, untreated polystyrene.Furthermore, these plates require that a single (pre-defined) attachmentchemistry be used in all wells, limiting the types of biomolecules thatcan be immobilized, as well as their orientations.

The present method may be used to produce chemically functionalizedmicrotitre plates that can allow multiple covalent immobilizationchemistries to be performed on a single plate for bioassay applications.Microtitre plates processed using this methodology may have versatileand predictable functionality and may possess surface properties thatenable the user to tailor the immobilization chemistry to suit thebiomolecule of interest.

Plasma treatments are a fine-tunable, dry alternative to commonly usedwet methods for the modification of polymeric surfaces (Davies et al.,“Argon plasma treatment of polystyrene microtiter wells. Chemical andphysical characterisation by contact angle, ToF-SIMS, XPS and STM”Colloids and Surfaces A: Physicochemical and Engineering Aspects, 174,287-295 (2000); Weikart et al., “Modification, Degradation, andStability of Polymeric Surfaces Treated with Reactive Plasmas” J. Polym.Sci. Part A: Polym. Chem., 38, 3028-3042 (2000); Guruvenket et al.,“Plasma surface modification of polystyrene and polyethylene” AppliedSurface Science, 236, 278-284 (2004)). Plasma treatment reduces thegeneration of hazardous chemical wastes with less degradation androughening of the material than wet chemical treatments. The advantageof plasma-based surface engineering is derived from the delivery ofenergetic ions and unique reactive species, which produces chemicalmoieties at the surface. Complex surface chemistries can be attained bysimply varying the gas that is used (e.g., Ar, N₂, O₂, H₂O, CO₂, NH₃),which provides the opportunity to endow the surface with specific andnon-specific performance features (Lane et al., “Surface treatments ofpolyolefins” Progress in Organic Coatings, 21(4), 269-284 (1993).Indeed, plasmas have been used to improve wettability, hydrophobicity,and biocompatibility of a wide variety of polymers (Garrison et al.,“Glow discharge plasma deposited hexafluoropropylene films: surfacechemistry and interfacial materials properties” Thin Solid Films,352(1-2), 13-21 (1999); Grace et al., “Plasma Treatment of Polymers”Journal of Dispersion Science and Technology, 24(3), 305-341 (2003);Hegemann et al., “Plasma treatment of polymers for surface and adhesionimprovement” Nuclear Instruments and Methods in Physics Research SectionB: Beam Interactions with Materials and Atoms Ionizing Radiation andPolymers, 208, 281-286 (2003); Lopez et al., “Immobilization of RGDpeptides on stable plasma-deposited acrylic acid coatings for biomedicaldevices” Surface and Coatings Technology PSE 2004, 200(1-4), 1000-1004(2005)).

Although energetic ions promote the various surface chemistries that aredesired, they can also be problematic. Consider that ions produced inconventional plasma discharges will impact the surface with energies onthe order of 10-50 eV, which exceeds the binding energies of polymericmaterials and can produce excessive surface roughening and etching andpromote the incorporation of non-native elements well below the surface.In addition, conventional discharges produce large densities of excitedspecies, leading to a significant flux of photons at the surface. Thesephotons can penetrate several hundred nanometers into the surface,inducing subsurface chemical changes that may not be beneficial. Thecombination of these effects likely lead to the well-known ageingbehavior in plasma modified polymers (Haidopoulos et al.,“Angle-Resolved XPS Study of Plasma-Deposited Polystyrene Films afterOxygen Plasma Treatment” Plasma processes and polymers, 5(1), 67-75(2008)), which could lead to relaxation to pre-treatment conditions.

In the disclosed method, an electron beam generated plasma is used, withthe ability to selectively alter the top few nanometers of a material'ssurface without changing the bulk properties, generating reactivechemical moieties that are incorporated at, rather than deposited on,the surface of a polymer. The properties of the plasmas used in thismethod include the delivery of ions with energies below the bindingenergy of polymeric materials and a low production of photons. Theelectron temperature (or the average electron energy) determines theproduction species and also the energy of ions incident to adjacentsurfaces. In conventional discharges, plasma electrons are energized viathe application of electric fields, producing a distribution of electronenergies with an average energy in the range of 2-10 eV. Only a smallfraction of the electrons are energetic enough to ionize the gas so mostof the electron energy is spent to excite the gas, producing largedensities of molecular fragments and excited species, which decay viaphoton emission. Moreover, the electron temperature determines theplasma's potential and thus determines the energy at which ions leavethe plasma. In electron beam generated plasmas, a high-energy (≈keV)electron beam is used to ionize the gas and the cross section forelectron-impact ionization at keV energies can be orders of magnitudehigher than the excitation cross sections. Thus, these sources generatea lower relative fraction of excited species that produce photons. Theelectron temperature in these plasmas is lower due to the absence of theelectric fields that are used in discharges. The resulting electrontemperatures are gas dependent but range between about 0.3 and 1 eV.Thus, incident ion energies are at or near the bond strength of mostpolymeric materials. These characteristics result in low etch rates,small changes in surface morphology, and generally induces chemicalmodifications limited to the top few nanometers of the polymer surface.

The method described herein consists of three steps that employ both dryand wet chemical processing of microtitre plates to yield a customized,multi-site covalent bioimmobilization platform for biomedical andbiological applications. The method combines the electron beam plasmasource described above to introduce surface hydroxyl (OH) with secondarysurface modification step using reactive end functionalizedorganosilanes. Multifunctional silane coupling agents combined withcovalent crosslinkers of various functionalities and spacer lengthsensures the stable attachment and functionally active biologicalcompounds. The three steps can be used in tandem to create microtitreplates with versatile and predictable functionalities that enable theuser to tailor the immobilization chemistry to suit the biomolecule ofinterest. Each step is described in detail in the following sections.

In the first step, an electron beam generated plasma is used tointroduce pendant hydroxyl groups onto the surface of an organicpolymer. The plasma generating apparatus may be that disclosed in Megeret al., U.S. Pat. No. 5,874,807. FIG. 1 schematically illustrates anexample plasma-generating apparatus. This apparatus uses a linear hollowcathode to produce a sheet-like electron beam. The shape of the beam canbe maintained by a magnetic field. One typical set of beam dimension islength of 50 cm, width of 25 cm, and thickness of 1 cm.

Organic polymers may have a carbon-based backbone. Any organic polymermay be used including polystyrene microtitre wells or a polymer, such aspolystyrene or polyethylene, spin-coated onto a substrate. This plasmadiffers notably from discharge plasmas, which are created by applyingelectric fields to a gas volume. In electron beam generated plasma, ahigh energy electron beam is injected into the gas, which ionizes,dissociates, and excites the gas. The resulting plasma differs fromdischarge plasmas in species generation and spatial evolution. Moreover,the inherently lower electron temperature results in a lower plasmapotential and thus the kinetic energy of ions impacting adjacentsurfaces is lower. Suitable gases for generating the plasma include, butare not limited to, argon, oxygen, carbon dioxide, dry air, humid air, agas with water vapor, or a mixture thereof. The treated polymer is thenexposed to air or other gas containing oxygen and hydrogen. Depending onthe gasses used, the hydroxyl groups may be generated by either theplasma treatment or the exposure to air.

When the plasma gas is argon, one set of suitable operating conditionsare within +/−10% of: gas pressure of 90 mTorr, treatment time of 20minutes, electron beam energy of 2 keV, electron beam current density of4 mA/cm², electron beam pulse width of 2 ms, electron beam pulsefrequency of 50 Hz. Other suitable conditions using different gases aredescribed by Lock et al. “Surface Composition, Chemistry, and Structureof Polystyrene Modified by Electron-Beam-Generated Plasma” Langmuir, 26,8857-8868 (2010). Generally, increasing the treatment time or gaspressure increases the total ion flux to the surface, which can increasethe concentration of hydroxyl groups on the surface. However, anexcessive ion flux may cause an increase in etching and favor chainscission over crosslinking as the dominant mechanism, reducing the yieldof hydroxyl groups. Example 2 shows the results of such variance.

For the second step in the process, plasma-treated microtitre platesdisplaying the necessary pendant surface hydroxyls are covalentlymodified with an organosilane coupling agent possessing a functional orreactive group at one end and one or several alkoxy or chloro groups atthe other. Suitable general organosilane formulas are R—SiX₃ andR′—CH₂)_(n)—SiX₃. R and R′ contain the functional or reactive group.Each X is an independently selected chemical group with the proviso thatat least one X is chloro, fluoro, bromo, halo, or alkoxy group. Thevalue n is a positive integer, including but not limited to integersfrom 1 to 25. Covalent attachment of the organosilane to theplasma-treated microplate surface yields a functionalized surface withpendant functional or reactive group capable of being used in asubsequent covalent coupling reaction. Given the large number ofpossible linking covalent methodologies used in subsequent steps, anyorganosilane terminating in any of the following functional or reactivegroups (R or R′) can be potentially used:

-   -   Functional groups:        -   Amine—can be coupled to amines, thiols, carboxyls        -   Thiol—can be coupled to amines, thiols, carboxyls        -   Aldehyde—can be coupled to amines        -   Carboxyl/nitrobenzyl-protected carboxyl groups—can be            coupled to amines    -   Reactive groups:        -   Glycidoxy (epoxy/epoxides)—can couple to thiols, hydroxyls            (pH-dependent)        -   Thiocyano, Isocyanate—can couple to hydroxyls, amines        -   Succinimidyloxy- or succinimidyl ester—can couple to amines        -   aryl azides/azido groups—non-selective linkage            (photoactivatable)        -   Hydrazine/hydrazide—can couple to aldehydes, oxidized            carbohydrates        -   Alkyl halide, benzyl halide, α-halo acetyl—can couple to            sulfhydryls        -   Maleimide—can couple to sulfhydryls

Silanes possessing these terminal groups are commercially available, butothers terminating in additional functional or reactive moieties canalso be used. For the work here, thiol- and amine-terminated silanes(MPTES and APTES) were incubated with plasma-treated plates, resultingin covalent functionalization with thiol and primary amine moieties,respectively, that are further modified with thiol or amine specificcrosslinkers (FIGS. 2A and 2C). The results support the suggestion thatmultiple chemistries may be carried out on a single plate.

More than one different organosilane compounds may be used on the samemicrotitre plate, using different compounds in different wells. Thisallows for the use of different chemistries for biomolecule attachment.The present method can result in the reproducible formation of amonolayer of the organosilane compound on the polymer surface, with thefunction or reactive groups primarily oriented away from the surface.This can be spatially favorable for attachment of the biomolecules.Other methods can cause a buildup of a thicker layer of the organosilanecompound in random orientations. The monolayer can also result in uniqueactivities of the biomolecules not found on other microtitre plates.

The third step in the process entails covalent attachment ofbiomolecules to the silane-functionalized surface, which may take placeas a single step (e.g., addition of a biomolecule to asilane-functionalized surface with a reactive terminal group; additionof a pre-activated protein) or as multiple steps (e.g., addition of acrosslinker, followed by the biomolecule). Suitable biomoleculesinclude, but are not limited to, peptides, antimicrobial peptides,carbohydrates, lipids, antibodies, and proteins. These single- ormulti-step methods for covalent attachment of biomolecules to the silanefunctionalized surfaces may be accomplished by numerous methods,including but not limited to the following methods.

(1) Addition of bi- or tri-functional chemical crosslinkers, withsimultaneous or subsequent incubation with biomolecule. A plethora ofhomo- and heterobifunctional crosslinkers are available commercially.These compounds will possess reactive groups on each end (e.g.,maleimide, N-hydroxysuccinimidyl (NETS) ester) that are designed to forma covalent adduct with functional groups such as thiols and amines,respectively. Examples of bifunctional linkers include GMBS(maleimide/NHS ester), BS3 (NETS ester/NHS ester), PMPI(maleimide/isocyanate), sulfo-HSAB (NETS ester/[photoactivatable] phenylazide), and EMCH (maleimide/hydrazide). Also considered included in thisgroup of chemical crosslinkers are the carbodiimides; this category ofmolecules mediates coupling between between carboxyls and primary amines(or hydrazides), resulting in a zero-length linkage and no addition of a“spacer”.

(2) Use of light to activate a terminal photoactivatable group on thecovalently bound silane, with simultaneous or subsequent incubation withbiomolecule. Several examples of photoactivatable silanes exist that canbe activated using ultraviolet or visible light: in general, the highlyreactive intermediates (e.g., nitrenes) formed upon photolysis willcreate covalent links non-specifically with amine, sulfhydryl, carbonyl,carboxyl, and reactive hydrogen groups. An alternate example ofphoto-induced modification of a silane terminal group is the uv-mediatedconversion of aldehyde-terminated monolayers of triethyoxysilylundecanalto carboxy-terminated layers (Hozumi et al., “Formation of aldehyde- andcarboxy-terminated self-assembled monolayers on SiO₂ surfaces” SurfInterface Anal., 40, 408-411 (2008)); these newly-formed carboxylmoieties can then presumably be used to attach amine-containingbiomolecules as described in (1) above.

(3) Use of pH or hydration to activate terminal reactive moietiespresent on the covalently attached silane and simultaneous or subsequentincubation with biomolecule. A number of silanes terminated in reactivegroups such as maleimide, NETS-ester, and isocyanate which will, underspecific conditions, react with thiols, amines, and/or hydroxyls,respectively. Epoxide-terminated silanes (e.g.,(3-glycidoxypropyl)trimethoxysilane) are a special case wherein the pHof the reaction dictates the specificity of covalent bond linkage;formation of thioether links (attachment to thiols) can occur at lowerpHs than secondary amine linkages (attachment to amines) or ether links(attachment to hydroxyls)

(4) Incubation with a modified biomolecules possessing a reactive groupspecific to the silane's terminal moiety. One example is incubation ofan activated protein such as maleimide- or glutaraldehyde-activatedhorseradish peroxidase (obtained from Pierce) with plate coated with athiol- or amine-terminated silane, respectively.

More than one of these crosslinking methods may be used on a singleplate, with different methods used in different wells. This is the casewhether the wells contain the same or different organosilane compounds.

A key feature of using this method to modify microtitre plates is that,after initial plasma treatment to create surface hydroxyls, the choiceof how each biomolecule can be attached is decided by the user. Morespecifically, by utilizing different combinations of silanes andbiomolecule-linking chemistries, the user can attach differentbiomolecules in discrete locations on the microplate (i.e., differentwells) using an appropriate covalent attachment chemistry tailoredspecifically to the individual biomolecules; likewise, one biomoleculecan be covalently immobilized in multiple wells using differentattachment chemistries.

The coupling of both dry and wet chemical methods to yield functionalsurfaces may have a number of advantages. Plasma treatment produces auniform, functionalized surface that is amenable to broad range ofchemistries. The wet chemical processing steps are variable such that, alarge variety of biomolecules can be immobilized. Moreover, the methodsdescribed are addressable in terms of location—an advantage not affordedby commercial activated plates which utilize a single attachmentchemistry for all wells. That is, while plasma can functionalize theentire plate, different wet chemistries can be applied to differentwells, allowing a wider variety of biomolecules to be immobilized, usingwider variety of covalent attachment chemistries.

An additional potential advantage is the lowered cost involved inmanufacturing these custom-tailored plates. Manufacture of microtitreplates used in clinical diagnostics, monitoring of foodstuffs, biotech,vaccine and therapeutics development, and many other areas of basic andapplied research represents a multi-billion dollar enterprise. Thismethod allows the user to save significant costs in tailoring theattachment chemistry to the biomolecule: standard hydrophobic microtitreplates costing˜$3 each are converted to plates capable of covalentattachment chemistry; commercial plates capable of similar covalentattachment chemistries typically cost $15 to $25 each.

The following examples are given to illustrate specific applications.These specific examples are not intended to limit the scope of thedisclosure in this application.

EXAMPLE 1

Materials—Microfluor I and NUNC Immobilizer Amino 96-well microtiterplates were obtained from Thermo Fisher Scientific (Rochester, N.Y.).Rabbit anti-lipid A (Escherichia coli) IgG was purchased from AbDSerotec (Raleigh, N.C.). Antimicrobial pep-tides (AMPs) cecropin A,cecropin P1, melittin, and cecropin A(1-8)-melittin (1-18) amide werereceived from American Peptide Company, Inc. (Sunnyvale, Calif.). Cy3monofunctional N-hydroxysuccinimidyl ester, 4-maleimidobutyric acidN-hydroxysuccinimide ester (GMBS), and bis(sulfosuccinimidyl) suberate(BS³) were purchased from Amersham-Pharmacia (Piscataway, N.J.).Lipopolysaccharide (LPS) from Salmonella typhimurium,(3-mercaptopropyl)triethoxysilane (MTPES),(3-aminopropyl)triethoxysilane (APTES), phosphate buffered saline (PBS),pH 7.4, potassium hydroxide, Tween-20, bovine serum albumin (BSA),dimethyl sulfoxide (DMSO), and methanol were purchased fromSigma-Aldrich (St. Louis, Mo.).

Preparation of Fluorescently Labeled IgG and LPS—Rabbit IgG and LPS wereconjugated with Cy3 monoreactive dye following manufacturer'sinstructions. Briefly, one mg IgG or LPS in 50 mM sodium borate, pH 8.5was incubated with one packet of Cy3 monoreactive dye, previouslydissolved in 25 μL anhydrous DMSO. After 1 h incubation at roomtemperature, the labeled IgG and LPS were purified from unincorporateddye by gel filtration on BioGel P-10 and BioGel P-2, respectively(BioRad, Hercules, Calif.). The labeled biomolecules were stored in thedark at 4° C. until use. The molar ratios of dye to labeled speciesranged from 1.7 to 3.5 for antibodies and 1.1 to 1.4 for LPS.

Microtitre plates were exposed to pulsed, electron beam generated plasmaproduced in argon, which activates the plate surface. OH groups areformed on the surface upon exposure to air, most likely resulting fromdissociative adsorption of H₂O. The experimental apparatus is discussedat Leonhardt et al., IEEE Trans. Plasma Sci., 33, 783 (2005). The systemvacuum was maintained by a 250 L/s turbo pump, with a base pressure of5×10⁻⁶ Torr. The operating pressure was achieved by introducing argon(purity >99.9999%), through mass flow controllers and by throttling thepumping speed using a manual gate valve. The electron beam was producedby applying a −2 kV pulse to a linear hollow cathode for a selectedpulse width and duty factor. The emergent beam passed through a slot ina grounded anode and was then terminated at a second grounded anodelocated further downstream. Beam spreading from collisions with thebackground gas was suppressed by a coaxial magnetic field (150 G)produced by a set of external coils. The microtiter plates were placedon a 10.2 cm diameter stage located at 2.5 cm from the nominal edge ofthe electron beam. The stage was held at ground potential and roomtemperature.

Microtiter Plate Silanization and Immobilization of IgG—NUNC MicrofluorI 96-well microtiter plates were rinsed with methanol prior tosilanization. Microwells were covered with 100 μL of 2% silane solution(MPTES or APTES) prepared in methanol adjusted to pH 4 by addition ofseveral drops acetic acid. After 30 min of incubation under nitrogen,plates were removed, rinsed three times in methanol, and dried.Cross-linking was performed by covering microwells with 100 μL of 1.0 mMGMBS in absolute ethanol and incubating for 30 min. Alternatively, 100μLof 50 mM BS³ in 10 mM phosphate buffer, pH 6.0, was used. Plates wererinsed thrice with 200 μL deionized water and dried; 1 μg/mL, 3 μg/mL,or 10 μg/mL Cy3-labeled antibody (in PBS) was then added into eachmicrowell and allowed to incubate for 2 h at room temperature withgentle agitation; each concentration was patterned in quadruplicate.Each microwell was washed three times with 250 μL of PBS with 0.005%Tween 20 (PBST). One-hundred microliters of PBS was added to eachmicrowell and the plate was read on a Tecan Safire microplate reader.

Immobilization of Peptides and LPS Binding Assay—Microwells were coveredwith 100 μL of 2% mercaptosilane solution prepared in acidic methanoland incubated for 30 min under nitrogen. Cross-linking was accomplishedby covering microwells with 100 μL of 1.0 mM GMBS in absolute ethanoland incubated for an additional 30 min. One-hundred microliters ofantimicrobial peptide solution (250 μg/mL in PBS) was added to each welland allowed to incubate at room temperature for 2 h with gentleagitation; each peptide was patterned in quadruplicate. For detection,after three washes with PBST, each well received 100 μL ofCy3-conjugated LPS and was incubated for 2 h at room temperature withgentle agitation. After this time, each microwell was washed three timeswith PBST; 100 μL PBS was added to each microwell and the plate was readon the Tecan microplate reader. For comparison of binding patterns,peptides were also immobilized onto standard glass slides as previouslydescribed using MPTES and GMBS (Kulagina et al., Anal. Chim. Acta, 575,9 (2006); Taitt et al., in Peptide Microarrays: Methods and Protocols;Cretich, M. Chiari, M., Eds.; Springer: New York, 2009; p 233).Cy3-labeled LPS was applied to the glass-immobilized peptides andincubated for 2 h, prior to washing with PTST and imaging using aPackard ScanArray Lite confocal microarray scanner; data were extractedfrom images using QuantArray microarray analysis software. To emphasizethe differences in the patterns of binding, as well as to account fordisparity in measurements obtained on the two instruments (microarrayscanner, micro-titer plate reader), data from both microtiter plates andglass slides were normalized with respect to LPS-melittin binding.

Water Contact Angle (WCA) Measurements—Goniometry was performed using astatic sessile drop technique on micro-titer plate fragments. Contactangle measurements were performed at room temperature using a goniometer(AST Products, Inc.), equipped with a microsyringe to control volume ofthe liquid drop (2 μL). Four water drops were placed at differentlocations on each substrate surface. Eight contact angle measurements(each side of one water drop) were averaged and corresponding standarddeviation were calculated for each plate fragment.

X-ray Photoelectron Spectroscopy (XPS)—Surface elemental and chemicalstate analyses were performed on a K-Alpha X-ray photoelectronspectrometer (Thermo Scientific). This instrument is equipped with amicrofocusing monochromator (Al—Kα X-ray source, 1486.6 eV), which wasoperated at a spot size of 400 μm. Analyzer pass energies of 200 and 25eV respectively were used for elemental survey and chemically sensitivenarrow scan spectra. The detection angle was 0° , which provides averageanalyzed depth down to 10 nm. K-Alpha's charge compensation system wasused during the analysis, utilizing very low energy electrons and argonions to prevent any localized charge build-up. Spectra were referencedto the main C is peak at 285.0 eV and quantified using Scofieldsensitivity factors. The high-resolution elemental spectra were fittedusing a commercial XPS analysis software package Unifit as previouslydescribed (Lock et al., Langmuir, 26, 8857-8868 (2010)). Briefly, themultiple-component fitting in the C is region always started from thelowest binding energy component and its full width at half-maximum wasallowed to vary. A convolution of Gaussian and Lorentzian line shapeswas assumed for individual peaks, following the line shapeparametrization. To produce consistent fits of minor C 1s components,their widths were constrained to the FMHW of the first peak and theirpositions were assigned as follows C—CO₂ 285.7 eV, C—O 286.6 eV, CdO287.6 eV, and O—CdO 289 eV. A linear combination of Shirley and linearfunctions with consistent parameters was used to model the background.

Atomic Force Microscopy (AFM)—The polymer surface morphology wasexamined using an atomic force microscope (Nanoscope III, VeecoMetrology, Santa Barbara, Calif.) operated in tapping mode. Surfaceimages were obtained from 5×5 μm² scans at resolution of 256×256 pixelsand scan rate of 1 Hz. For a quantitative evaluation of the topographychanges, root-mean-square (rms) roughness was calculated from thesurface height data z_(i) using

$R_{q} = \left\lbrack {\frac{1}{N}{\sum\limits_{i = 1}^{N}{{z_{i} - \overset{\_}{z}}}^{2}}} \right\rbrack^{1/2}$where z is the mean height.

Characterization of Polystyrene Microtiter Plates after PlasmaTreatment—As expected, the untreated polystyrene was hydrophobic (WCA of95.93±4.57° and showed carbon as the predominant constituent (C1s92.78±1.43 at. %. FIGS. 3A-B show the survey spectra of microtitreplates, which indicates the presence of oxygen, and FIGS. 4A-B show thehigh resolution C1s spectrum, which indicates the detail the oxygenbinding at the surface. Table 1 shows the relative atomic composition inFIGS. 3A-B. Table 2 shows the relative concentrations of the speciesused in the fitting in FIGS. 4A-B. After plasma treatment, thepolystyrene surface exhibited a significant increase in oxygenconcentration (from 5.3 to 16.26 at. %; P<0.005), with trace levels ofsilicon (<0.5 at. %) detected as well. The high-resolution C1s spectradetailed the plasma-mediated formation of oxygen-based functionalgroups, including C—O (hydroxyls, ethers) and CdO (epoxy, aldehydes,ketones); the presence of these moieties resulted in an increase insurface hydrophilicity, as confirmed by the 2-fold reduction of the WCAto 48.68±4.37°. The majority of oxygen incorporated into the polymersurface was in the form of singly bonded oxygen; however, C—O wasindistinguishable from C—OH in the high-resolution spectra. Gas-phasechemical derivatization analysis (Alexander et al., “Effect ofelectrolytic oxidation upon the surface chemistry of type A carbonfibres: III. Chemical state, source and location of surface nitrogen”Carbon, 34(9), 2093-1102 (2008)) based on fluorine labeling usingtrifluoroacetic anhydride indicated that nearly all of the C—O/C—OHbonds designated in the high resolution C1s spectra were attributed toC—OH bonds. In contrast to reactive gases, plasmas created in inert gasdo not directly introduce functional groups to the surface. However,they do promote surface chemistries via the breaking of bonds,generating reactive sites (dangling bonds) where functional groups canattach (Lock et al., Plasma Process Polym., 6, 234 (2009)); theincorporation of oxygen occurs at those locations upon sample exposureto air after plasma treatment. In this case, hydroxyl groups were mostlikely formed because of the dissociative adsorption of water uponexposure to ambient air (Petrat et al., Surf Interface Anal., 21, 274(1994); Petrat et al., Surf Interface Anal., 21, 402 (1994)). Aspreviously noted, the incident ion energy is small in electron beamgenerated plasmas and thus the creation of reactive sites is notaccompanied by significant changes in morphology as shown in FIGS. 5(a)and (c) (P>0.5 data not shown). Thus, incorporation of functionalgroups, and not surface roughness, is responsible for the observedchanges in water contact angle. As will be discussed later, this uniqueplasma surface treatment promotes uniform silane layer formation andsmall peptide immobilization.

TABLE 1 Elements PS reference (at. %) Plasma treated PS (at. %) C1s95.96 69.63 O1s 3.27 21.47 Si2p 0.77 3.9 Zn2p 1.04 Ca2p 1.04 Na1s 0.51Cl2p 1.34 F1s 1.07

TABLE 2 C1 components Quantity (at. %) C—C, C—H 84.65 C—O (C—OH) 10.67C═O 6.03 O—C═O 1.66

A broad range of operating conditions was tested; the various plasmasources and operating conditions are shown in Table 3. Specifically, thepulse width, duty factor, ambient pressure, and total exposure timeswere varied. However, successful silanization of the polystyrenesurface, as determined by attachment of a fluorescently labeled protein,was limited to electron beam produced plasmas and a narrow range ofoperating conditions (FIGS. 2A-D). The most successful result,determined by the efficiency of covalent biomolecule attachment, were 2ms pulse widths, 10% duty factor, 90 mTorr operating pressures, and 2minutes exposure time. It is believed that the relative flux of ionsproduced under these conditions is important to producing the correctsurface conditions within the wells of the microtitre plates.

TABLE 3 Treatment RF DC HC Sample Pressure Time Power Offset VoltageDuty Assay number (mTorr) Gas (sec) (W) (V) (keV) (%) success C1-C3 90Ar 30 — — 2 10 No C4-C5 90 Ar 30 — — 2 10 No C6 90 Ar 60 — — 2 10 No C790 Ar 120 — — 2 10 Yes C10 90 Ar 300 — — 2 10 No C11 60 Ar 30 — — 2.4 10No C14 100 Ar 120 — — 2 10 No C15 110 Ar 120 — — 2 10 No C19 90 Ar 120 —— 2 20 No C20 50 Ar/O₂ 30 — — 2 10 No C21-23 90 Ar 120 — — 2 10 Yes

As noted, OH formation occurs after exposure to air. It is likely thatsimilar OH formation could be achieved by treating the plates withplasma produced in reactive backgrounds as well. Candidate gasbackgrounds would need to contain some amount of both oxygen andhydrogen such as, but not limited to combinations of O₂ and H₂, humidair, or H₂O or H₂O₂ vapors in a carrier gas. It is likely that thesetypes of gas backgrounds would also change the operating conditions ofthe plasma, compared to those described above.

Attempts to use other plasma sources yielded inferior and/orinconsistent results. The reasons for this could be that the range ofoperating conditions explored was not broad enough to yield a flux ofions comparable to those produced in electron beam plasmas. As notedabove, electron beam generated plasmas produce ion fluxes that arecharacterized by significantly lower kinetic energies. It is known thatthe energy of ions will depend on the collisionality of the sheathsformed at surfaces and that collisionality will depend on both theplasma density and operating pressure. Thus, one might be able toachieve comparable ion energies in any plasma source.

Characterization of Polystyrene Microtiter Plates afterSilanization—XPS, WCA, and AFM measurements were also used to evaluatethe efficiency and uniformity of silanization on plasma-treated anduntreated polystyrene surfaces. Because a mercaptosilane was used in thesilanization step, the presence of sulfur (—SH, binding energy (BE) at163 eV) and silicon (O—Si—C, BE at 102.6 eV) in the XPS survey spectraserved as indicators for silanization. The sulfur and silicon unique tothe silane were differentiated from trace element contaminants in thecommercial micro-titer plates; sulfur content/contamination from theplates was identified in its oxidized form—SO (BE at 168 eV) and most ofthe silicon was in the form of Si—O (BE at 103.3 eV) rather than O—Si—C(28).

The XPS results of the silanized, plasma-treated polystyrene surfaceshowed a significant increase in both sulfur and silicon content (≈4.0at. %) compared to the silanized, untreated polystyrene (<1.3 at. %;P<0.005) Table 4 shows the relative atomic concentrations (%) andstandard deviation (values represent means of threedeterminations±standard deviations) of the measured elements, watercontact angles (WCA), and roughness (rms) values of mercaptotosilanizedpolystyrene microtitre plate before and after argon plasma treatment.These results indicated that the silanization efficiency was enhanced bythe plasma-induced modification of the polymer surface. WCA of theplasma-treated silanized plate also indicated that silanizationoccurred; the WCA value changed from 48.68±4.37° (before silanization)to 60.05±2.08° (after silanization) (P<0.025). The increased WCA valueis attributed to the hydrophobic alkyl chain in MPTES and is consistentwith literature values (Darian et al., Biosens. Bioelectronics, 24, 1744(2009)). A moderate (but not significant) change in WCA uponsilanization was also observed with plates not treated with plasma(P>0.05). However, the final WCA values observed with untreated,silanized plates showed a larger standard deviation, indicating moreheterogeneous surface and presumably less efficient silane deposition.The uniformity of silane deposition was further evaluated by atomicforce microscopy. Even though the average roughness values ofplasma-treated and untreated silanized polymer plates were approximatelythe same, the standard deviations in both cases were different; asmaller standard deviation was observed after plasma treatment.Furthermore, a significant globular formation on the surface on theuntreated plate is also apparent (cf. FIG. 5A and with FIG. 5B). Silaneaggregation was observed by other research groups as well (Darian etal., Biosens. Bioelectronics, 24, 1744 (2009)). It should be noted thatafter electron beam plasma treatment, only a few aggregates wereobserved (FIG. 5D).

TABLE 4 substrate MPTES C O Si (102.3 eV) S (163 eV) WGE (deg)^(a) rmsMT plate − 92.8 ± 1.4  5.3 ± 1.0 1.2 ± 0.6 0.3 ± 0.3 95.9 ± 4.6 7.0 ±1.7 MT plate + 82.7 ± 0.6 12.3 ± 1.0 0.5 ± 0.1 1.3 ± 0.1  81.9 ± 10.18.3 ± 2.4 MT plate, − 80.9 ± 0.4 16.3 ± 0.3 0.4 ± 0.1 ND^(b) 48.7 ± 4.47.1 ± 1.7 plasma treated MT plate, + 72.2 ± 1.1 16.5 ± 1.3 4.3 ± 0.3 4.0± 0.3 60.1 ± 2.1 7.9 ± 0.5 plasma treated ^(a)Value represents mean ±standard error of four averages at different locations. ^(b)ND, notdetected.

Biomolecule Immobilization Efficacy—To demonstrate the suitability ofthe system for versatile, covalent immobilization of biomolecules toplasma-modified plates, two possible schemes were examined (FIG. 6). Inscheme one in FIG. 6, plasma-modified microtiter plates werefunctionalized with APTES with the intention of presenting surface aminegroups. The APTES-treated microwell surfaces were subsequently reactedwith an amine-reactive homobifunctional cross-linker (BS³), which allowsthe covalent attachment of Cy3-conjugated IgG. In scheme two in FIG. 6,MPTES was used to form a silane monolayer presenting surface thiolgroups. The MPTES-treated microwell surfaces were then reacted with aheterobifunctional cross-linker (GMBS) followed by the covalentattachment of Cy3-conjugated IgG. In both schemes, untreated polystyrenemicrotiter plates served as controls. On the basis of the two differentimmobilization schemes presented, BS³, which contains two amine-reactiveNHS groups, is expected to react only with APTES. GMBS, which displaysboth an NHS group and a maleimide group that react with amine andsulfhydryl groups, respectively, is expected to react with both APTESand MPTES; however, only the MPTES-GMBS combination is expected promoteefficient protein conjugation.

As mentioned above, FIGS. 2A-D represent the mean net fluorescencevalues of attached Cy3-IgG (at 1.0, 3.0, and 10 μg/mL) usingimmobilization Schemes one and two. To fully demonstrate the ability ofthe plasma-silane cross-linking methodology to enable multipleimmobilization strategies on the same plate, APTES-BS³, MPTES-GBMS, andcombinations thereof (i.e., the “wet” chemistry) were tested on the sameplasma-treated 96-well plates, or untreated plates as a control.Fluorescent signals on the plasma-treated plate were shown to besignificantly higher than the untreated plate (compare FIGS. 2A and Cwith FIGS. 2B and D, respectively; P<0.05). Only low fluorescent signalswere detected at even the highest protein concentration tested on theuntreated plate; these levels, corresponding to <0.02 pmol/well, arepresumably due to background levels of adsorption onto the microtiterplate surface, and are well-below published saturation values for IgGadsorbed to polystyrene (0.35-0.5 pmol/cm² (Rossier et al., “Langmuir,16, 8489 (2000); Salonen et al., Immunol. Methods, 30, 209 (1979);Nygren et al., Immunol. Methods, 101, 63 (1987)). Although someimmobilization was observed when GMBS was used to cross-link theantibody to APTES-treated plates, Cy3-IgG immobilization on APTES-coatedsurfaces was much more efficient when BS³ was used (FIG. 2A);GMBS-mediated linking of Cy3-IgG to APTES surfaces may have occurred byreaction with free thiols present on native or partially denatured IgGs,or potentially from low levels of maleimide reaction with excess amineson IgG surfaces (Brewer et al., Anal. Biochem., 18, 248 (1967)). On theother hand, bioimmobilization on MPTES coated surfaces was greatest inthe presence of GMBS cross-linker (FIG. 2C). Although the MPTES-BS³demonstrated higher relative fluorescence, it was comparable to thecontrol samples containing no cross-linker.

IgGs and many other large biomolecules can be immobilized using a widerange of substrates and linking procedures with minimal impact onbinding activity. However, smaller biomolecules such as peptides,saccharides, and small proteins are highly sensitive to surfacemorphology and orientation when immobilized (Ngundi et al., Sens. Lett.,5, 621 (2007); Inamori et al., Anal. Chem., 80, 643 (2008); Disney etal., Chem. Biol., 11, 1701 (2004); Kulagina et al., Anal. Chim. Acta,575, 9 (2006); Kulagina et al., Anal. Chem., 77, 6504 (2005)). Todemonstrate that the described chemistry could be applied to this lattercategory of biomolecules, a series of naturally occurring antimicrobialpeptides were utilized as model ligand-binding molecules. Thesebiomolecules are capable of binding to a large variety of microbialcells with different affinities, which makes them useful in detectionassays (Kulagina et al., Anal. Chem., 77, 6504 (2005)); the ability todistinguish between different categories of microbial cells based ontheir patterns of binding to different peptides can be used for rapidand broad-spectrum screening (Kulagina et al., Sens. Actuators B, 121,150 (2007); Kulagina et al., Anal. Chim. Acta, 575, 9 (2006); Taitt etal., in Peptide Microarrays: Methods and Protocols; Cretich, M. Chiari,M., Eds.; Springer: New York, 2009; p 233). Previous studies have shownthat when immobilized onto glass slides using an amine-directedimmobilization strategy (MPTES treatment, followed by GMBS, analogous toscheme two), the peptides displayed unique patterns of binding toGram-positive and -negative bacteria (Shriver-Lake et al., In Biosensorsand Biodetection: Methods and Protocols: Electrochemical and MechanicalDetectors, Lateral Flow and Ligands for Biosensors; Rassoly, A., Herold,K. E., Eds.; Springer: New York, 2008; p 419, North et al., Anal. Chem.,82, 406 (2009)) and to the Gram-negative biomarker lipopolysaccharide,LPS (FIG. 7, white bars). Surprisingly, we observed very differentpatterns of LPS binding when utilizing commercial, preactivatedmicrotiter plates (Immobilizer amino, maleic anhydride; FIG. 7, stripedbars). Although both types of commercial plates use amine-directedchemistry (as did the glass slides), the differences in reactive groups,overall surface characteristics, and linker lengths appear to affect theimmobilized peptides' ability to bind the target LPS, presumably byaltering peptide orientation, density, freedom of movement, and/or thenumber and identity of linked amino acid side chains. However, whenplasma-treated plates were processed using analogous chemistry as usedon the glass slides (plasma-treatment→silanization withMPTES→cross-linking with GMBS), the binding pattern observed (FIG. 7,gray bars) was similar to that of the gold standard glass slides. Theability to transition an immobilization methodology directly from oneplatform (silanized glass slides) to another (plasma-treated, silanizedplates), with minimal alteration of binding specificity, illustrates apotential advantage conferred by the method described here.

Comparison between Plasma-Treated and Commercial Microtiter Plates—Theissues of biomolecule denaturation, desorption, and/or loss ofbiomolecule activity that could occur when biomolecules are immobilizedthrough physisorption have been recognized and not, surprisingly anumber of commercial microtiter plate are available in preactivated formfor covalent attachment of biomolecules (amine-directed immobilization:Immobilizer Amino and maleic anhydride-functionalized;sulfhydryldirected immobilization: maleimide-functionalized). Theefficacy of biomolecule immobilization (using immobilization scheme two)on plasma-treated, silanized plates was compared with those ofcommercial microtiter plates utilizing both physisorption andamine-directed, covalent attachment strategies. Protein immobilizationefficiency (fluorescence of immobilized Cy3-IgG) was determined for eachkind of plate before and after a stringent detergent wash (buffer with0.5% Tween-20; FIG. 8). Consistent with data presented in FIGS. 2A-D,plasma-treated plates exhibited significantly higher fluorescent signalthan untreated plates (≈2-fold increase) where immobilization hasoccurred via physisorption (“Microfluor I Untreated”; P<0.005). Asexpected, the difference between the plasma-treated and untreated platesincreases dramatically (≈8-fold difference) after a detergent wash.Although covalent attachment of IgG to the commercial preactivatedplates (Maleic anhydride, NUNC Immobilizer Amino) preserved a greaterproportion of the attached biomolecules, the overall binding efficiencyof plasma-/silane-treated plates was still superior (P<0.01).

EXAMPLE 2

The microtitre plates were placed on a 10.2 cm diameter stage located at2.5 cm from the nominal edge of the electron beam. The stage was held atground potential and room temperature. The total gas flow rate was heldconstant at 50 sccm. The experimental conditions are summarized in Table5.

TABLE 5 Exp. # Pressure (mTorr) Time (min) Duty factor (%) 1 60 0.5 10 290 2 10 3 95 2 10 4 90 1 10 5 90 5 10 6 90 2 20

As variation in pressure has the greatest influence on the production ofplasma species—charged particles, metastables and photons, it isexpected to produce the most pronounced difference in oxygenincorporation. The highest degree of oxygen incorporation was observedat a pressure of 90 mTorr (exp #2, Tables 5, 6) (O/C=0.3) resulting inthe highest polar component of the surface energy as well. The mostlikely mechanism of polystyrene modification in argon is the formationof carbon-centered polymer free radicals (P●), which preferentiallycrosslink. However, chain scission and formation of polyenes are alsopossible (Knobloch, Angew. Makromol. Chem., 176-177, 333 (1990)). Higherpressures (exp #3, Table 5) did not improve the surface chemicalcomposition. In fact, the O/C ratio, C—O group concentration and γ_(p)decreased. This might be due to an excessive ion flux, which may causean increase in etching and favor chain scission over crosslinking as thedominant mechanism. The surface roughness decreased at all pressures,which might be caused by ablation of low molecular weight fragmentsresiding on the polymer surface.

TABLE 6 C—C(H)^(a), π − Exp # O/C C—CO₂ ^(a) C—O^(a) C═O^(a) COO^(a)π*^(a) γ_(p) ^(b) γ_(d) ^(b) γ_(tot) ^(b) RMS^(c) bioefficiency Ref 0.0392.61 1.58 0 0 5.81 0 40 40 19 − 1 0.27 89.84 5.12 2.94 1.08 1.01 24 3256 11 − 2 0.30 86.07 8.24 4.04 1.65 0 26 38 64 12 + 3 0.20 73.03 5.563.24 1.05 0 10 43 53 12 − 4 0.22 77.74 3.14 0.33 0 0.9 20 36 56 11 − 50.23 87.53 7.8 2.85 1.02 0.8 19 39 58 16 − 6 0.14 81.32 6.17 1.56 0 0 1340 53 ND^(d) − ^(a)Concentration of functional groups was measured inat. %. ^(b)Surface energy was measured in mJ/m². ^(c)RMS roughness wasmeasured in nanometers (nm). ^(d)ND means not determined.

Variations in both treatment time and duty factor at a fixed pressureled to similar conclusions. Increase in treatment time from 1 to 2minutes (exps #4 and 2, Tables 5, 6) resulted in higher O/C ratios,increased amount of oxygen functionalities (C—O, C═O, COO groups) andimproved polar component of the surface energy with no change in surfaceroughness. However, when the polystyrene substrate was treated for 5minutes (exp #5, Tables 5, 6) the O/C ratio decreased and surfaceroughness increased. These results suggested that prolonged plasmaexposure increases etching, increases surface roughness, and promoteschain scission and not crosslinking. Increase in duty factor (exp #2 and#6) did not increase the oxygen incorporation. In fact, it resulted indecrease of O/C ratio as well as a decrease of the polar component ofthe surface energy by a factor of two. Although the reasons are notclear, it appears that reducing the time between pulses does not promotethe incorporation of OH-groups.

Covalent bioimmobilization was observed only at a pressure of 90 mTorr,treatment time of 2 minutes and 10% duty factor (exp. #2 Table 6, FIG.9). At this condition the increase in fluorescent signal, signifyingspecific biomolecule attachment, was 26-fold above the backgroundlevels. These results are in direct correlation to the presence ofoxygen functional groups on the polystyrene surface. That is, when theoxygen functionalities concentration on the polymer surface wasoptimized, it allowed for efficient silanization and thus specificbiomolecule immobilization occurs.

Obviously, many modifications and variations are possible in light ofthe above teachings. It is therefore to be understood that the claimedsubject matter may be practiced otherwise than as specificallydescribed. Any reference to claim elements in the singular, e.g., usingthe articles “a,” “an,” “the,” or “said” is not construed as limitingthe element to the singular.

What is claimed is:
 1. An article made by a method comprising: treatinga microtitre plate comprising an organic polymer and comprising aplurality of wells with an electron beam-generated plasma; exposing thetreated polymer to air or an oxygen- and hydrogen-containing gas;wherein the treating and exposing generates hydroxyl groups on thesurface of the polymer; reacting the surface with an organosilanecompound having a chloro, fluoro, or alkoxy group and a functional orreactive group that is less reactive with the surface than the chloro,fluoro, or alkoxy group; and covalently immobilizing a biomolecule tothe functional or reactive group or a reaction product thereof.
 2. Thearticle of claim 1; wherein the reaction product is made by reacting theorganosilane compound with a crosslinker; and wherein the biomolecule iscovalenlly immobilized to the crosslinker.
 3. The article of claim 2,wherein the organosilane compounds in at least two different wells areeach reacted with a different crosslinker.